The following references are cited in this Background Section:
1. Schauer, R., Adv Carbohydr Chem Biochem (1982) 40: 131 to 234. PA1 2. Corfield, A. P., et al, "Sialic Acids, Chemistry, Metabolism and Function", pp 5-50, Schauer, R., ed., Springer-Verlag, New York (1982). PA1 3. Hakomori, S., TIBS (1984) 453 to 459. PA1 4. Feizi, T., et al, TIBS (1985) 24 to 29. PA1 5. Paulsen, H., et al, Carbohydr Res (1986) 146: 147 to 153. PA1 6. Sabesan, S., et al, J Amer Chem Soc (1986) 108: 2068 to 2080. PA1 7. Paulsen, H., et al, Carbohydr Res (1984) 125: 47 to 64. PA1 8. Paulsen, H., et al, Carbohydr Res (1985) 144: 205 to 229. PA1 9. Ogawa, T., et al, Eur Pat Appl Pub No 146090, Jun. 26, 1985. PA1 10. Ogawa, T., et al, Tetrahedron Lett (1986) 27:5739 to 5742. PA1 11. Pozsgay, V., et al, Carbohydr Chem (1987) 6: 41 to 55. PA1 12. Paulsen, H., et al, Carbohydr Res (1985) 137: 63 to 77. PA1 13. Ogawa, T., et al, Eur Pat Appl Pub No. 166442, Jan. 2, 1986. PA1 14. Paulson, J. C., et al, Pure Appl Chem (1984) 56: 797-806. PA1 15. Loomes, L. M., et al, Nature (1984) 307: 560-563.
Sialic acid glycosides are known to occur in a wide variety of biological materials .sup.1,2 in the form of gangliosides and complex oligosaccharides attached to proteins. These are present in bodily fluids and on cell surfaces. Sialic acid-containing structures have been shown to be important for the attachment of viral particles to tissues and protection of proteins from proteolysis. They are known to be higher in concentration.sup.3,4 in sera of cancer patients as opposed to normal individuals; they also occur on the tissues of cancer patients. Specifically structures having as terminal tetrasaccharides 19-9 and sialo-X moieties are related to the cancerous state. Assays taking advantage of this association are described in U.S. Pat. No. 4,471,057; and antibody production to tumors bearing these haptens in U.S. Pat. No. 4,172,124.
In order to detect, quantify and study the tetrasaccharides (and their precursors and biosynthesis) it is advantageous to obtain them and their antibodies in practical amounts. The availability of these moieties from nature through isolation is tedious and results in limited quantities of material that must be purified for further use. Also, material that is obtained through isolation does not provide for useful modified structures, such as synthetic antigens or immunoadsorbents.
An alternative to isolation of such interesting structures to provide well-defined materials for the study of biological actions is chemical synthesis. The chemical synthesis of sialosides in high anomeric purity and reasonable yields has remained a difficult challenge for chemists in the recent past.sup.5,6.
There has been moderate success in chemically preparing sialosides with 2-6 linkages, however most reaction conditions with various substrates give anomeric mixtures (.alpha./.beta. equals near 1/1).sup.7,8,9,10. A wide variety of reaction conditions has been reported, including variation of substrate alcohol, catalyst, and solvent. The result of these reactions is wide variation of overall yield of sialosides (10-80%) but generally consistent .alpha./.beta. ratios of near 1/1 with few exceptions.sup.7,11. Such mixtures are tedious and difficult to separate to obtain the desired alpha-sialoside.
The reported methods for forming a glycosidic linkage between the two position of sialic acid and the three position of galactosides (2-3 linkage) and derivatives of these, have been even less successful.sup.5. Overall yields of sialosides (.alpha. and .beta.) are consistently lower and anomeric purity is poor. Again a wide variety of alcohols, catalysts and solvents have been used in these attempts. As it has been shown.sup.7,12 that there is great variation obtained (both in anomeric specificity and overall yield) with various acceptors, donors and reaction conditions in the formation of 2-6 linkages, extrapolation from these results to the formation of 2-3 linkages in a meaningful way is difficult and uninstructive. The danger of such comparisons is well known to the skilled chemist.
All but five reported examples of sialoside synthesis of higher oligosaccharides use a step-wise synthetic strategy. The examples of "block synthesis" used to produce higher sialosides involving the use of a 2-6 block show limited versatility, or poor anomeric specificity.sup.8,10,13.
The one reported example of the synthesis of a 2-3 block suffers from the same problems more severely. This block is produced through the reaction of a sialoside derivative with a 3,4 diol of a disaccharide which results in the 2-3 linkage in 17% yield with an .alpha./.beta. ratio of 0.4. This block has not been used for the synthesis of larger oligosaccharides to effect an intersugar linkage.sup.13.
The one consistent factor in all strategies for the synthesis of higher sialosides is the use of a methyl ester as the temporary blocking group for the acid moiety of the sialosyl halide. Use of this group would seem sensible as it provides the necessary blocking while conferring minimal steric interference adjacent to the carbon through which the glycosidic linkage is to be formed, and it is believed that the inherent steric restriction around carbon two of ketoses is, in part, also responsible for the increased production of undesirable unsaturated products during glycosylation of sialic acid derivatives.
The use of a methyl ester derivative of the sialyl halogenose results in limitation of subsequent use of the product oligosaccharide for the formation of synthetic antigens and immunoadsorbents which are among the objects of this invention. This limitation is due to the desirability of being able to easily deblock a synthetic sialoside, including its acid group, while maintaining an ester group present in a linking arm, attached to the oligosaccharide, for subsequent activation to allow coupling of the sialoside to proteins and insoluble carriers. Such coupling is achieved for most oligosaccharides through attachment of a synthetic oligosaccharide to an amino or carboxylic acid group on a protein carrier. The strategies of coupling to carboxylic acid groups in proteins are precluded, as it would be commonplace to use an amino-terminated linking arm. This would result in undesirable self-polymerization of the now carboxylic unprotected synthetic oligosaccharide. Therefore, terminally derivatized acid linking arms which, by the nature of the ester or other derivative, can be chemically differentiated from the ester used to block the sialyl acid group are preferred, and these derivatives must be persistent through the total deblocking of the sialoside.
There are few reports in the prior art of the preparation of any synthetic sialyl oligosaccharide antigens and immunoadsorbents or the properties of these. There are no reports, to our knowledge, of higher (more than one or two different sugar residues) synthetic sialyl oligosaccharide antigens or immunoadsorbents.